The present invention relates generally to microbial transporter proteins and, more specifically, to novel 2,5-diketo-D-gluconic acid (2,5-DKG) permeases.
Adequate intake of ascorbic acid, or vitamin C, is recognized as an important factor in maintaining health. To ensure adequate intake of ascorbic acid, the chemical is now added to many foods, drinks and cosmetic products, and is also sold as a direct vitamin supplement. To meet the commercial demand for ascorbic acid, there is a need to develop more efficient processes for its production.
Although there are a number of alternative methods of producing ascorbic acid, one of the least expensive and most ecologically sound methods is biofermentation. Bacterial strains have now been engineered to express all of the enzymes required for the stepwise conversion of an inexpensive sugar source, such as D-glucose, to a stable precursor of ascorbic acid, 2-keto-L-gulonic acid (2-KLG) (see U.S. Pat. No. 5,032,514 and references therein). 2-KLG can be readily converted to ascorbic acid by chemical or enzymatic procedures.
FIG. 2 shows schematically the enzymatic reactions that take place in the bioconversion of D-glucose to 2-KLG. As shown in FIG. 2, the enzymatic reactions that lead from D-glucose, to D-gluconic acid, to 2-keto-D-gluconic acid (2-KDG), to 2,5-diketo-D-gluconic acid (2,5-DKG), take place at the surface of the bacterial cell. 2,5-DKG must then enter the cell in order for its enzymatic conversion to 2-KLG.
Much effort has been expended in increasing the efficiency of the enzymatic reactions involved in 2-KLG production. For example, U.S. Pat. No. 5,032,514 describes methods for increasing 2-KLG production by reducing metabolic diversion of 2,5-DKG to products other than 2-KLG.
Increasing uptake of 2,5-DKG by a bacterial strain suitable for biofermentation could be advantageous in increasing 2-KLG production. Expressing additional copies of an endogenous 2,5-DKG permease, or expressing an exogenous 2,5-DKG permease with superior properties, could increase uptake of 2,5-DKG. However, to date, no 2,5-DKG permease has been identified or characterized that could be used in this manner.
Therefore, there exists a need to identify and characterize nucleic acid molecules encoding 2,5-DKG permeases, so that permeases with advantageous properties can be used in the commercial production of ascorbic acid and in other important applications. The present invention satisfies this need and provides related advantages as well.
The invention provides an isolated nucleic acid molecule encoding a polypeptide which has 2,5-DKG permease activity. In one embodiment, the isolated nucleic acid molecule contains a nucleotide sequence having at least 40% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOS:1, 3, 5, 7, 9 and 11. In another embodiment, the isolated nucleic acid molecule contains a nucleotide sequence which encodes a polypeptide having at least 40% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10 and 12.
Also provided are vectors and cells containing isolated nucleic acid molecules encoding polypeptides having 2,5-KDG permease activity. In one embodiment, the cells are bacterial cells selected from the genera Pantoea and Klebsiella.
The invention also provides methods of identifying and isolating nucleic acid molecules encoding polypeptides which have 2,5-DKG permease activity. Also provided are methods of enhancing 2-KLG production, by expressing the nucleic acid molecules of the invention in suitable bacterial cells.
Further provided are isolated polypeptides having 2,5-DKG permease activity, and immunogenic peptides therefrom. The invention also provides antibodies specific for such polypeptides and peptides.